The specific aims of the proposed research are to a) elucidate the influence of dUMP as a determinant of the responsiveness to 5-fluorouracil (FUra) of human colorectal adenocarcinoma xenografts maintained in immune-deprived mice, b) evaluate the role of the formation of non-covalent ternary complexes between 5-fluorodeoxy-uridylate (FdUMP) or deoxyuridylate (dUMP), thymidylate synthetase (TS) and folic acid and its derivatives (5-CH3FH4, 5-CHOFH4, CH FH4 and FH2) in increasing both the degree and duration of inhibition of TS selectively in tumors, c) examine the rate of metabolism of radiolabelled folates in tumors and in normal tissues, d) evaluate the toxicity and antitumor activity of agents that increase the endogenous pool of dUMP (FUra or methotrexate (MTX)), or produce FdUMP, (FUra, 5-fluoro-2 feet-deoxyuridine) in combinations that have demonstrated a basis for selectivity in the preceding studies, and e) elucidate a possible basis for selectivity in the scheduling of MTX prior to FUra. Levels of dUMP will be determined in cytosols from tumors and normal tissues both before and after treatment of mice with either FUra or MTX. The formation of non-covalent complexes in tissue cytosols will be determined from the addition of folic acid and its derivatives at various concentrations in the presence of endogenous cofactor and either FdUMP or dUMP with isolation on nitrocellulose filters, and will be related to the residual activity of TS determined in vitro. The concentrations of folates achievable in tissues after injection or infusion and their rate of metabolism will be determined by HPLC analysis of tissue powders prepared under liquid nitrogen immediately after excision. The toxicity and antitumor activity of combinations of agents will be examined in vivo. Compounds to be used to attempt to selectively reduce 5-phosphoribosyl-1-pyrophosphate (PRPP) in normal tissues after the administration of MTX in vivo include folic acid and its derivatives, purine nucleosides, bases and thymidine; PRPP will be assayed in vitro. The long term objectives are to derive selective therapy for the treatment of human colon carcinoma.